When cells are subjected to oxidative stress during inflammation and exposure to environmental pollutants, such as nitrogen dioxide or ozone, peroxidation of membrane lipids yields several reactive end products, among which 4-hydroxy-2-nonenal (HNE) is a major species. This highly electrophilic alpha, beta-unsaturated aldehyde can modify DNA and proteins to alter their functions and produce toxic effects implicated in the pathogenesis of many diseases, including cancer, Alzheimer's disease, and atherosclerosis. Though cysteine is known to be the primary target for HNE modification, much less is known about the specific protein targets that are modified by HNE or the precise sites of these modifications, which is critical information required to understand how HNE produces its cellular effects. The candidate proposes to perform quantitative analysis of protein targets and cysteine sites of HNE modification in the human proteome using advanced chemical proteomic methods. They will also structurally and functionally characterize reactive cysteines in select protein targets that are super-sensitive towards HNE modification, and validate their roles in regulating HNE-mediated signaling pathways under oxidative stress. The candidate anticipate that the proposed project will identify key HNE-reactive cysteine residues from various cellular pathways in the human proteome and provide molecular details on the role of HNE in modulating protein structure and function and, through doing so, illuminate mechanisms by which HNE confers changes in cell biology caused by oxidative stress. Public Health Relevance: When organisms are exposed to environmental pollutants, such as nitrogen dioxide or ozone, 4-hydroxy-2- nonenal (HNE) is a major product yielded from cellular oxidative stress that can modify proteins toxically to cause chronic inflammation and cancer. In order to understand the molecular details of toxicity conferred by HNE, the investigators will perform quantitative profiling of protein targets and sites of HNE modification in the human proteome using advanced chemical proteomic methods. They will also structurally and functionally characterize specific HNE-sensitive proteins involved in various cellular pathways responsive to oxidative stress.